Endocrine Abstracts

Brown adipose tissue (BAT) primarily performs non-shivering thermogenesis by metabolising lipids using mitochondrial uncoupling protein 1 (Ucp1). Differentiation of brown preadipocytes is essential for this function, driven by key transcription factors such as Pparg, which promotes lipid storage and sensitivity to insulin and β-adrenergic agonists. The Tubby protein, first identified by its mutant variant in mice for its extreme obesity and diabetes phenotype, is expressed in adipose tissue and has been linked to insulin sensitivity and resistance. Tubby contains a DNA-binding domain and prior work suggests Tubby is a potential transcriptional regulator during BAT maturation. To investigate Tubby’s role in brown adipocyte differentiation, we characterised its expression via western blotting as well as immunofluorescence analysis. RNA-sequencing analysis was performed on differentiating brown preadipocytes following Tubby siRNA knockdown to identify potential transcriptional targets, after 24 hours of differentiation. Selected potential target genes were validated by qRT-PCR. Tubby expression was found to be highest throughout the cell at 24 hours after initiation of differentiation, while also translocating to the nucleus, before decreasing in expression until fully matured. Analysis of the RNAseq data identified several essential BAT functional pathways affected by Tubby knockdown, including Ppar signalling and lipolysis. Specifically, expression of key genes such as Pparg and Fabp4, were significantly reduced following Tubby depletion (> 0.5 log2fold change in expression). This data indicates that Tubby is crucial for the appropriate transcriptional cascade required for brown adipocyte maturation. Our findings suggest that the protein Tubby acts as a transcription factor essential for the regulation of brown adipocyte differentiation, potentially through the direct or indirect control of master regulators like Pparg at an early stage in BAT differentiation. Further investigation is needed to fully characterise the DNA-binding activity of Tubby and its precise mechanism of action within the BAT differentiation pathway.